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Immobilized RNA switches for the analysis of complex chemical and biological mixtures 总被引:11,自引:0,他引:11
A prototype biosensor array has been assembled from engineered RNA molecular switches that undergo ribozyme-mediated self-cleavage when triggered by specific effectors. Each type of switch is prepared with a 5'-thiotriphosphate moiety that permits immobilization on gold to form individually addressable pixels. The ribozymes comprising each pixel become active only when presented with their corresponding effector, such that each type of switch serves as a specific analyte sensor. An addressed array created with seven different RNA switches was used to report the status of targets in complex mixtures containing metal ion, enzyme cofactor, metabolite, and drug analytes. The RNA switch array also was used to determine the phenotypes of Escherichia coli strains for adenylate cyclase function by detecting naturally produced 3',5'- cyclic adenosine monophosphate (cAMP) in bacterial culture media. 相似文献
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Peng Q Kong N Wang HC Li H 《Protein science : a publication of the Protein Society》2012,21(8):1222-1230
Synthetic/artificial protein switches provide an efficient means of controlling protein functions using chemical signals and stimuli. Mutually exclusive proteins, in which only the host or guest domain can remain folded at a given time owing to conformational strain, have been used to engineer novel protein switches that can switch enzymatic functions on and off in response to ligand binding. To further explore the potential of mutually exclusive proteins as protein switches and sensors, we report here a new redox-based approach to engineer a mutually exclusive folding-based protein switch. By introducing a disulfide bond into the host domain of a mutually exclusive protein, we demonstrate that it is feasible to use redox potential to switch the host domain between its folded and unfolded conformations via the mutually exclusive folding mechanism, and thus switching the functionality of the host domain on and off. Our study opens a new and potentially general avenue that uses mutually exclusive proteins to design novel switches able to control the function of a variety of proteins. 相似文献
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We present a self-consistent field approximation approach to the problem of the genetic switch composed of two mutually repressing/activating genes. The protein and DNA state dynamics are treated stochastically and on an equal footing. In this approach the mean influence of the proteomic cloud created by one gene on the action of another is self-consistently computed. Within this approximation a broad range of stochastic genetic switches may be solved exactly in terms of finding the probability distribution and its moments. A much larger class of problems, such as genetic networks and cascades, also remain exactly solvable with this approximation. We discuss, in depth, certain specific types of basic switches used by biological systems and compare their behavior to the expectation for a deterministic switch. 相似文献
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Tavva VS Dinkins RD Palli SR Collins GB 《The Plant journal : for cell and molecular biology》2006,45(3):457-469
The ecdysone receptor (EcR) has been used to develop gene switches for conditional regulation of transgene expression in plants and humans. All EcR-based gene switches developed to date for use in plants are monopartate and require micromolar concentrations of ligand for activation of the transgene; this has limited the use of these gene switches. We have developed a Choristoneura fumiferana ecdysone receptor (CfEcR)-based two-hybrid gene switch that works through the formation of a functional heterodimer between EcR and the retinoid X receptor (RXR) upon application of the chemical ligand methoxyfenozide. Methoxyfenozide is already registered for field use with an excellent safety profile, and it has potential as a gene switch ligand for applications in the field. The receptor constructs were prepared by fusing DEF domains (hinge region plus ligand-binding domain) of CfEcR to the GAL4 DNA-binding domain and EF domains (ligand-binding domain) of ultraspiracle from Choristoneura fumiferana (CfUSP) or RXR from Locusta migratoria (LmRXR), Mus musculus (MmRXR) or Homo sapiens (HsRXR) to the VP16 activation domain. These receptor constructs were tested for their ability to induce expression of the luciferase gene placed under the control of 5x GAL4 response elements and -46 35S minimal promoter in tobacco, corn and soybean protoplasts and in transgenic Arabidopsis and tobacco plants. By adopting the two-hybrid format, the sensitivity of the CfEcR gene switch has been improved from micromolar to nanomolar concentrations of methoxyfenozide. The sensitivity of the CfEcR + LmRXR two-hybrid switch was 25 to 625 times greater than the monopartate gene switch, depending on the plant species tested. 相似文献
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Tavva VS Palli SR Dinkins RD Collins GB 《Archives of insect biochemistry and physiology》2007,65(3):164-179
Genetic engineering of plants using transgenic technology is targeted to enhance agronomic performance or improved quality traits in a wide variety of plant species, and has become a fundamental tool for basic research in plant biotechnology. Constitutive promoters are presently the primary means used to express transgenes in plants. However, inducible gene regulation systems based on specific chemicals have many potential applications in agriculture and for enhancing the basic understanding of gene function. As a result, several gene switches have been developed. The ecdysone receptor gene switch is one of the best inducible gene regulation systems available, because the chemical, methoxyfenozide, required for its regulation is registered for field use. An EcR gene switch with a potential for use in large-scale field applications has been developed by adopting a two-hybrid format. In a two-hybrid switch format, the GAL4 DNA binding domain (GAL4 DBD) was fused to the ligand binding domain (LBD) of the Choristoneura fumiferana ecdysone receptor (CfEcR); and, the VP16 activation domain (VP16 AD) was fused to the LBD of Locust migratoria retinoid X receptor (LmRXR). The sensitivity of the CfEcR gene switch was improved from micromolar to nanomolar concentrations of ligand by using the CfEcR:LmRXR two-hybrid switch. In this report, we demonstrate the utility of CfEcR:LmRXR two-hybrid gene switch in functional genomics applications for regulating the expression of a Superman-like single zinc finger protein 11 (ZFP11) gene in both Arabidopsis and tobacco transgenic plants. 相似文献
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Rho proteins are small GTPases of the Ras superfamily that regulate a wide variety of biological processes, ranging from gene expression to cell migration. Mechanistically, the major Rho GTPases function as molecular switches cycling between an inactive GDP-bound and an active GTP-bound conformation, although several Rho proteins spontaneously exchange nucleotides or are simply devoid of GTPase activity. For over a decade, RhoGEFs and RhoGAPs have been established as the mainstream regulators of Rho proteins, respectively flipping the switch on or off. However, regulation by GEFs and GAPs leaves several fundamental questions on the operation of the Rho switch unanswered, indicating that the regulation of Rho proteins does not rely exclusively on RhoGEFs and RhoGAPs. Recent evidence indeed suggests that Rho GTPases are finely tuned by multiple alternative regulatory mechanisms, including post-translational modifications and protein degradation, as well as crosstalk mechanisms between Rho proteins. Here we review these alternative mechanisms and discuss how they alter Rho protein function and signaling. We also envision how the classic binary Rho switch may indeed function more like a switchboard with multiple switches and dials that can all contribute to the regulation of Rho protein function. 相似文献
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Lichen symbioses are defined as a symbiotic relationship between a mycobiont (generally an ascomycete) and one or more photobionts (green algae or/and cyanobacteria). It was proposed that cephalodia emancipation is an evolutionary driver for photobiont switch from chlorophyte to cyanobacteria. In this study we want to test the monophyly of cyanolichens and to measure the phylogenetic signal of the symbiotic relationship between cyanobacteria and a mycobiont partner in the lichen genus Pseudocyphellaria. This genus includes some species that have a chlorophyte as primary photobiont (and Nostoc in internal cephalodia), while others have only cyanobacteria. In a phylogenetic framework we measure the phylogenetic signal (or phylogenetic dispersion) as well as mapped photobiont switches performing stochastic character mapping. Results show that having cyanobacteria as main photobiont has a strong phylogenetic signal that follows a Brownian motion model. Seven clades in the phylogeny had an ancestor with cyanobacteria. Reversal to a green algae photobiont is rare. Several switches were estimated through evolutionary time suggesting that there was some flexibility in these traits along the phylogeny; however, close relatives retained cyanobacteria as main photobiont throughout the cyanolichen’s history. Photobiont switches from green algae to cyanobacteria might enhance ecotypical differentiation. These ecotypes could lead to several speciation events in the new lineage resulting in the phylogenetic signal found in this study. We give insights into the origin of lichen diversity exploring the photobiont switch in a phylogenetic context in Pseudocyphellaria s. l. as a model genus. 相似文献
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Sai Sumanjali Yarra Ganapathy Ashok Utpal Mohan 《Biotechnology and bioengineering》2023,120(4):932-952
Toehold switches are de novo designed riboregulators that contain two RNA components interacting through linear-linear RNA interactions, regulating the gene expression. These are highly versatile, exhibit excellent orthogonality, wide dynamic range, and are highly programmable, so can be used for various applications in synthetic biology. In this review, we summarized and discussed the design characteristics and benefits of toehold switch riboregulators over conventional riboregulators. We also discussed applications and recent advancements of toehold switch riboregulators in various fields like gene editing, DNA nanotechnology, translational repression, and diagnostics (detection of microRNAs and some pathogens). Toehold switches, therefore, furnished advancement in synthetic biology applications in various fields with their prominent features. 相似文献
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Cellular receptors can act as molecular switches, regulating the sensitivity of microbial proteins to conformational changes that promote cellular entry. The activities of these receptor-based switches are only partially understood. In this paper, we sought to understand the mechanism that underlies the activity of the ANTXR2 anthrax toxin receptor-based switch that binds to domains 2 and 4 of the protective antigen (PA) toxin subunit. Receptor-binding restricts structural changes within the heptameric PA prepore that are required for pore conversion to an acidic endosomal compartment. The transfer cross-saturation (TCS) NMR approach was used to monitor changes in the heptameric PA-receptor contacts at different steps during prepore-to-pore conversion. These studies demonstrated that receptor contact with PA domain 2 is weakened prior to pore conversion, defining a novel intermediate in this pathway. Importantly, ANTXR2 remained bound to PA domain 4 following pore conversion, suggesting that the bound receptor might influence the structure and/or function of the newly formed pore. These studies provide new insights into the function of a receptor-based molecular switch that controls anthrax toxin entry into cells. 相似文献
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Nucleotide-binding proteins are often used as molecular switches to control the assembly or activity of macromolecular machines. Recent work has revealed that such molecular switches also regulate the spread of some mobile DNA elements. Bacteriophage Mu and the bacterial transposon Tn7 each use an ATP-dependent molecular switch to select a new site for insertion and to coordinate the assembly of the transposition machinery at that site. Strong parallels between these ATP-dependent transposition proteins and other well-characterized molecular switches, such as Ras and EF-Tu, have emerged. 相似文献
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Protein conformational switches are ubiquitous in nature and often regulate key biological processes. To design new proteins that can switch conformation, protein designers have focused on the two key components of protein switches: the amino acid sequence must be compatible with the multiple target states and there must be a mechanism for perturbing the relative stability of these states. Proteins have been designed that can switch between folded and disordered states, between distinct folded states and between different aggregation states. A variety of trigger mechanisms have been used, including pH shifts, post-translational modification and ligand binding. Recently, computational protein design methods have been applied to switch design. These include algorithms for designing novel ligand-binding sites and simultaneously optimizing a sequence for multiple target structures. 相似文献
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J. Lu S. Gramoll J. Schmidt R. L. Calabrese 《Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology》1999,184(3):311-324
The motor program for heartbeat in the medicinal leech is produced by a central pattern generator that regularly switches
between two alternative coordination states. A pair of switch heart interneurons reciprocally alternate between rhythmically
active and inactive states to effect these switches. During spontaneous switches in the activity state of switch interneurons,
there was no correlation between the duration of a particular activity state and beat period, indicating that the timing networks
for the switch cycle and the beat cycle are relatively independent. Simultaneous recordings from two switch heart interneurons
showed that a perturbation in the electrical activity of one does not influence switching of the other and that there is no
synaptic interaction between them. Using voltage clamp, we characterized an L-like Ca2+ current (measured as Ba2+ currents), inactivating and non-inactivating K+ currents, a persistent Na+ current, and a hyperpolarization-activated inward current in switch interneurons. Dynamic clamp experiments show that “subtraction”
of an artificial switch leak conductance (described previously by Gramoll et al. 1994) from a switch interneuron when it is
in the inactive state causes it to display activity associated with the active state. We discuss how the switch leak conductance
may interact with the intrinsic currents of switch interneurons to control their activity state.
Accepted: 1 December 1998 相似文献
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S A Frank 《Proceedings. Biological sciences / The Royal Society》1999,266(1426):1397-1401
Trypanosoma brucei infects various domestic and wild mammals in equatorial Africa. The parasite's genome contains several hundred alternative and highly diverged surface antigens, of which only a single one is expressed in any cell. Individual cells occasionally change expression of their surface antigen, allowing them to escape immune surveillance. These switches appear to occur in a partly random way, creating a diverse set of antigenic variants. In spite of this diversity, the parasitaemia develops as a series of outbreaks, each outbreak dominated by relatively few antigenic types. Host-specific immunity eventually clears the dominant antigenic types and a new outbreak follows from antigenic types that have apparently been present all along at low frequency. This pattern of sequential dominance by different antigenic types remains unexplained. I use a mathematical model of parasitaemia and host immunity to show that small variations in the rate at which each type switches to other types can explain the observations. My model shows that randomly chosen switch rates do not provide sufficiently ordered parasitaemias to match the observations. Instead, minor modifications of switch rates by natural selection are required to develop a sequence of ordered parasitaemias. 相似文献
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Production of proteins and biochemicals in microbial cell factories is often limited by carbon and energy spent on excess biomass formation. To address this issue, we developed several genetic growth switches based on CRISPR interference technology. We demonstrate that growth of Escherichia coli can be controlled by repressing the DNA replication machinery, by targeting dnaA and oriC, or by blocking nucleotide synthesis through pyrF or thyA. This way, total GFP-protein production could be increased by up to 2.2-fold. Single-cell dynamic tracking in microfluidic systems was used to confirm functionality of the growth switches. Decoupling of growth from production of biochemicals was demonstrated for mevalonate, a precursor for isoprenoid compounds. Mass yield of mevalonate was increased by 41%, and production was maintained for more than 45 h after activation of the pyrF-based growth switch. The developed methods represent a promising approach for increasing production yield and titer for proteins and biochemicals. 相似文献
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A common problem in determining a number of important gait parameters is a convenient means for indicating foot/floor contact. Various foot switches have been used in many forms, but still an ideal, commercially available switch has yet to materialize. This note describes one method for constructing very thin, (1 mm), durable, switches that have worked well in the Gait Analysis Lab at Texas Scottish Rite Hospital. 相似文献
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This protocol describes a new and rapid isothermal reaction process designed to amplify and detect a specific DNA sequence in purified DNA extracted from cultured cells. The protocol uses a DNA nanomachine that comprises two molecular switches that function in concert to isothermally amplify and detect a DNA target. First, a molecular beacon detection switch is 'activated' only if a DNA target sequence is present. A DNA primer and DNA polymerase are used to lock the beacon in an activated conformation. Second, an amplification and signal-transduction switch is initiated following successful activation. A nicking endonuclease and the DNA polymerase are used to replicate the DNA target. Both switches operate simultaneously at 40 °C in a single reaction to rapidly generate multiple copies of the DNA target in a cyclic polymerization reaction. This protocol enables femtomole amounts of a DNA target to be reproducibly amplified and detected in <40 min. We demonstrate the successful use of this protocol in assays containing synthetic DNA components and purified DNA extracted from biological samples. 相似文献
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Chemical inducible gene regulation systems provide essential tools for the precise regulation of transgene expression in plants
and animals. Recent development of a two-hybrid ecdysone receptor (EcR) gene regulation system has solved some of the drawbacks
that were associated with the monopartate gene switch. To further improve the versatility of the two-hybrid EcR gene switch
for wide spread use in plants, chimeras between Homo sapiens retinoid X receptor (HsRXR) and insect, Locusta migratoria RXR (LmRXR) were tested in tobacco protoplasts as partners with Choristoneura fumiferana EcR (CfEcR) in inducing expression of the luciferase reporter gene. The RXR chimera 9 (CH9) along with CfEcR, in a two-hybrid
format gave the best results in terms of low-background expression levels in the absence of ligand and high-induced expression
levels of the reporter gene in the presence of nanomolar concentrations of the methoxyfenozide ligand. The performance of
CH9 was further tested in corn and soybean protoplasts and the data obtained was compared with the other EcR switches that
contained the wild-type LmRXR or HsRXR as EcR partners. In both transient expression studies and stable transformation experiments,
the fold induction values obtained with the CH9 switch were several times higher than the values obtained with the other EcR
switches containing LmRXR or HsRXR. The new CfEcR two-hybrid gene switch that uses the RXR CH9 as a partner in inducing reporter
gene expression provides an efficient, ligand-sensitive and tightly regulated gene switch for plants. 相似文献
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Wei Tang Jiaqi Sun Jia Liu Fangfang Liu Jun Yan Xiaojun Gou Bao-Rong Lu Yongsheng Liu 《Plant molecular biology》2014,84(4-5):443-454
Chemically inducible gene switches can provide precise control over gene expression, enabling more specific analyses of gene function and expanding the plant biotechnology toolkit beyond traditional constitutive expression systems. The alc gene expression system is one of the most promising chemically inducible gene switches in plants because of its potential in both fundamental research and commercial biotechnology applications. However, there are no published reports demonstrating that this versatile gene switch is functional in transgenic monocotyledonous plants, which include some of the most important agricultural crops. We found that the original alc gene switch was ineffective in the monocotyledonous plant sugar cane, and describe a modified alc system that is functional in this globally significant crop. A promoter consisting of tandem copies of the ethanol receptor inverted repeat binding site, in combination with a minimal promoter sequence, was sufficient to give enhanced sensitivity and significantly higher levels of ethanol inducible gene expression. A longer CaMV 35S minimal promoter than was used in the original alc gene switch also substantially improved ethanol inducibility. Treating the roots with ethanol effectively induced the modified alc system in sugar cane leaves and stem, while an aerial spray was relatively ineffective. The extension of this chemically inducible gene expression system to sugar cane opens the door to new opportunities for basic research and crop biotechnology. 相似文献